CHDI’s 7th Annual HD Therapeutics Conference took place February 27 – March 1, 2012, in Palm Springs, California. This unique conference series focuses on drug discovery and development for Huntington’s disease, and draws participants and speakers from the biotech and pharmaceutical sectors as well as academia and research institutions. The conference is intended as a forum where all participants can share ideas, learn about new disciplines, network with colleagues and build new collaborative partnerships. We are indebted to all of the conference speakers, and especially grateful to those who are able to make their presentations available here for a wider audience.
- Systems Approaches to Neurodegenerative Diseases and the Emergence of Transforming Techniques Lee Hood, MD, PhD
- The Application of Systems Biology to the Discovery and Development of HD Therapeutics Keith Elliston, PhD
- A Genetic Foundation for Integrating Large Datasets to Define HD Pathogenesis James F. Gusella, PhD
- 3D Brain Atlases of Model Animals: Synapses, Neurons and Brain Compartments Hanchuan Peng, PhD
- RNA Quality Control Pathways as Potential Therapeutics Targets in HD Melissa J. Moore, PhD
- A Role for Hunting in Post-Transcriptional Gene Expression Naoko Tanese, PhD
- Defining Posttranslational Modifications in Huntingtin Lisa M. Ellerby, PhD
- The Role of Posttranslational Modifications in Clearance of Mutant Huntingtin Dimitri Krainc, MD, PhD
- Genetics Based Discovery of Huntingtin in Post-Translational Modification Marcy MacDonald, PhD
- Advances in RNAi Therapies for Huntington’s Disease Beverly L. Davidson, PhD
- Antisense Based Therapeutics for the Treatment of Huntington’s Disease C. Frank Bennett, PhD
- Zinc Finger Proteins: New Possibilities for HD Research and Therapy Steve Zhang, PhD
- Use of Biomarkers to Bridge Preclinical and Clinical Studies of Huntingtin Suppression by RNAi Therapeutics William F. Kaemmerer, PhD
- Silencing Sheep – Exploring Safety and Efficacy of Viral Based Gene Therapy in a Large Animal Model Neil Aronin, MD
- Phosphodiesterases (PDEs) as CNS Targets Christopher Schmidt, PhD
- cGMP-hydrolyzing PDEs are Therapeutics Targets for Huntington’s Disease Vahri Beaumont, PhD
- Neuropharmacology of Novel Kynurenine 3-monooxygenase Inhibitors in Animal Models of Huntington’s Disease Ladislav Mrzljak, MD, PhD
- Selective mGluR5 Antagonists as Therapies for Movement and Neurodevelopmental Disorders Graeme Bilbe, PhD
- A Refreshing Look at Huntington’s Disease Clinical Trials Cristina Sampaio, MD, PhD
- TRACK-HD: Final Analysis and a New Beginning Sarah Tabrizi, MD, PhD
- Huntington’s Disease Diagnosis: Moving from an Event to a Spectrum Mark Guttman, MD, FRCPC
- Changing Prevalence of HD: Altered Demographics and its Implications Michael Hayden, MD, PhD
Systems Approaches to Neurodegenerative Diseases and the Emergence of Transforming Techniques
Lee Hood, MD, PhD – Institute for Systems Biology
The challenge for biology in the 21st century is the need to deal with the incredible complexity of biological systems and disease. The challenge in understanding disease complexity is that of deciphering the operations of dynamic biological networks and molecular machines. One approach to deciphering this complexity is to generate enormous amounts of information about the systems of interest—across the multiple scales of biological information (DNA, RNA, protein, interactions, etc.) and then integrate these data into predictive models of health and disease.
I will focus on our efforts at taking a systems approach to disease—looking at a neurodegenerative (prion) disease, We published a few years ago a study on prion disease that has taken more than 6 years to integrates 6 different types of data and lay out the principles of a systems approach to disease. From these studies and the others come a clear understanding of some of the principal opportunities systems biology brings to medicine and the study of disease. These include: powerful new approaches to delineate disease mechanisms, making blood a window into health and disease, the stratification of diseases into different subtypes so as to be able create an impedance match with effective drugs, a new approach to the identification of drug targets and the ability to analyze multiple organ responses to disease.
I will also discuss the emerging technologies that will transform biology and medicine over the next 10 years—e.g., next generation DNA sequencing and its applications to human genome sequencing, targeted mass spectrometry, microfluidic protein chips, new approaches to proteincapture agents, single-cell analyses and the use of induced pluripotential cells to understand development, disease mechanisms and stratify disease.
It appears that systems medicine, together with emerging technologies and the development of powerful new computational and mathematical tools will transform medicine over the next 5-20 years from its currently reactive state to a mode that is predictive, personalized, preventive and participatory (P4). I will describe the nature of P4 medicine and its societal implications for healthcare.
The Application of Systems Biology to the Discovery and Development of HD Therapeutics
Keith Elliston, PhD – CHDI
Systems biology aims to describe and understand the operation of complex biological systems to give insight into the fundamental governing principles of the disease and, ultimately, to apply this knowledge to guide drug discovery and improve health. While the proximal cause of Huntington’s disease has been well known for some time – the expanded triplet repeat in the huntingtin gene – the molecular mechanisms that manifest in the disease are still unclear. The nascent systems biology effort at CHDI is applying state-of-the-art techniques and tools to define the underlying molecular mechanisms of disease in HD patients to inform drug discovery, and to identify sensitive and appropriate animal- and cellbased models, new targets for therapeutic intervention, and biomarkers to assess mechanisms of disease and therapeutic response in the clinic. Systems approaches to biology rely upon coherent large-scale gene, protein and metabolite datasets (‘omics’) to develop computational models of disease that enable efficient hypothesis generation and testing, expediting discovery cycles. This marriage of large-scale empirical biology with advanced computational analysis and modeling promises to streamline and accelerate the discovery of therapeutics for HD. The goals, strategy, organization, and progress of the CHDI systems biology effort will be presented and discussed.
A Genetic Foundation for Integrating Large Datasets to Define HD Pathogenesis
James F. Gusella, PhD – Massachusetts General Hospital
Huntington’s disease is triggered by the presence of an expanded CAG repeat that is expressed, as polyglutamine in the huntingtin protein, with biochemical consequences throughout the lifespan though its effects usually become apparent clinically only in mid-life. Human genetic studies involving thousands of individuals have established that the rate of HD pathogenesis leading to neurologic onset is largely determined by the length of a single HD gene CAG repeat expansion but is also modified by other, as yet unidentified genetic factors. This finding provides the foundation for two broad types of investigations in which the integration of large datasets can support unbiased approaches for defining the pathogenic process and identifying critical factors that can alter it. In ongoing collaborative efforts, genome-wide association and DNA sequencing strategies are being pursued to allow integration of DNA variation in large numbers of HD subjects with a large set of phenotypic measures, beginning with age at motor onset. These studies aim to identify additional genetic factors that impact on the pathogenic process and result in alteration of the ultimate outcome measure. However, they also “book-end” in both function and time the intervening dynamic of biochemical and physiological processes that connect the cause of pathogenesis with its clinical consequence. This offers the opportunity to fill the intervening gap with molecular measures on the same sets of individuals to define the pathogenic process by integrated analysis of varied data types, guided by knowledge of the genetic underpinnings of disease in each individual. In particular, the strong mathematical relationship between CAG repeat length and rate of pathogenesis offers a reliable filter with which to extract relevant alterations in gene expression, microRNA expression, chromatin modification, protein expression, protein modification, metabolomics, clinical phenotype, etc., from large but inherently noisy datasets. The goal of this “system genetics’ strategy is to better define and model in an integrated manner the network of changes that lead from the initial mutation to the clinical disease, providing both clues to interventions that could alter this network and measures with which to test them.
Presentation not available
Hanchuan Peng, PhD – Howard Hughes Medical Institute
We built 3D digital neuron atlases for model animals including C. elegans, fruit fly, dragon fly, and mouse. In these models we quantitatively describe the wiring of neurite structures, projection and potential connectivity patterns of neurons, and distribution of synapses. For instance, we build a 3D map of the spatially stereotyped neurite tracts throughout a fruit fly’s brain, and reconstruct an initial fly brain wiring diagram how neurites connect and project across a complete set of 64 brain compartments. To produce this informative atlases, we developed a pipeline of image analysis and informatics tools to register high-resolution 3D laser scanning microscopic images accurately, followed by 3D image-visualization assisted neuron reconstruction methods (Vaa3D-Neuron) to digitize the morphology and location of neurite structures. We also demonstrate the usefulness of our approach for complicated mammalian (mouse) brains by quantitatively measuring the synapses visualized using the newly developed mouse-GRASP labeling technique. Our methods can be applied to a variety of brain structure-function studies.
RNA Quality Control Pathways as Potential Therapeutics Targets in HD
Melissa J. Moore, PhD – University of Massachusetts Medical School
Although research in the HD field has traditionally focused on expanded poly-Gln toxicity, recent work has indicated that expanded CAG repeats can also exert neuronal toxicity at the RNA level. Further, it is now known that CAG repeats can be translated in vivo in all three reading frames to generate poly-Ser and poly-Ala in addition to poly-Gln. While the extent to which RNA and poly-Ser/poly- Ala toxicity contribute to HD in humans is not yet established, specific removal of mutant HD RNA would have the advantage of eliminating both RNA and protein toxicities. One auspicious approach that recently entered large animal trials is specific knock down of mutant HD RNA by allele-specific RNAi. Yet because allele-specific RNAi relies on SNP heterozygosities between mutant and wild type HD alleles, it may not be applicable in all cases. Therefore, other means of silencing the mutant allele need to be explored. One idea is to harness one of the endogenous RNA quality control (QC) pathways. In particular, mRNA QC pathways that detect and eliminate stalled translation complexes may hold promise as targets whose upregulation could specifically down regulate mRNAs containing expanded CAG repeats.
A Role for Hunting in Post-Transcriptional Gene Expression
Naoko Tanese, PhD – New York University
We recently reported a new role for the HD protein huntingtin (Htt) in post-transcriptional gene regulation and maintenance of processing bodies / neuronal RNA granules (PNAS 2008:105,10820; JBC 2010:285,13142). Endogenous Htt was found to co-localize and co-traffic with mRNA in dendrites. An emerging body of evidence suggests regulated transport and local translation of mRNAs in neurons play a critical role in establishing their connectivity. Our findings implicate normal Htt in these important dynamic processes in neurons; it is possible that mutant Htt perturbs them in some way, contributing to the HD pathogenesis. We hypothesize that Htt is associated with a subset of mRNAs in neuronal granules and regulate transport and local translation of these mRNAs in response to synaptic activity.
Our previous study used non-neural cell lines to purify and identify Htt-associated proteins. To better understand the functions of Htt in the brain, we immunopurified Htt from mice expressing endogenous FLAG-Htt (provided by Dr. Scott Zeitlin), and identified proteins and RNA selectively associated with wild type and mutant Htt. Previously unreported Htt interactions with Myo5a, Prkra, Gnb2l1, Rps6, G3bp2, and Syt2 were confirmed by immunoblotting. Gene ontology analysis of Htt-associated proteins revealed a statistically significant enrichment for proteins involved in RNA processing and translation among other categories. Wild type and mutant Htt co-localized with the stress granule protein component Tia1 in metabolically stressed cultured cells. We also found cosedimentation of Htt with polysomes in cytoplasmic mouse brain extracts dependent on the presence of intact ribosomes. Together, these data support a role for Htt in protein translation. We think our new findings on Htt will lead to its previously undiscovered role in pathways that regulate gene expression at the post-transcriptional level.
Defining Posttranslational Modifications in Huntingtin
Presentation not available
Lisa M. Ellerby, PhD – The Buck Institute for Age Research
Polyglutamine expansion in the huntingtin protein is the cause of Huntington’s disease. A potential disease-modifying therapy would be one that causes the mutant Htt protein itself to no longer be toxic to cells. One approach is to determine which post-translational modifications in the context of mutant Htt that abolish toxicity. Post-translational modifications (PTMs) are rapid, effective and reversible ways to regulate the stability, proteolysis, localization, function, toxicity and protein interactions of Htt. A number of laboratories have explored how specific PTMs affect some of these events. We have developed methods to identify and quantify PTMs in huntingtin using an unbiased approach with mass spectrometry. This approach has identified novel sites of phosphorylation, acetylation and oxidation. The role of post-translational modifications in the biology and pathogenesis of Huntington’s disease will be discussed as well as potential approaches to target these PTMs for drug discovery.
The Role of Posttranslational Modifications in Clearance of Mutant Huntingtin
Dimitri Krainc, MD, PhD – Massachusetts General Hospital
In eukaryotic cells, acetylation is among the most common covalent modifications and ranks similar to the important master switch phosphorylation. The correlation between histone acetylation and increased transcription has been known for many years, but acetylases are now being identified to modify a number of non-histone proteins. Acetylation can affect many cellular functions including protein-protein interactions, microtubule dynamics, splicing, mRNA function, protein localization, metabolism, protein stability and aging. We have previously shown that acetylation of mutant huntingtin facilitates clearance of the mutant protein via macroautophagy. Here we examined the mechanisms of acetylation-mediated clearance of mutant Htt in more detail and tested whether inhibition of specific HDACs plays a role in the process. Specifically, we found that genetic or pharmacological inhibition of a subset of HDACs leads to increased acetylation and clearance of mutant huntingtin. This clearance occurs primarily via acetylation-dependent ubiqutination suggesting across talk between these posttranslational modifications. Together, our results suggest that isoform-specific HDAC inhibitors would be potentially useful to increase acetylation and clearance of mutant huntingtin.
Genetics Based Discovery of Huntingtin in Post-Translational Modification
Marcy MacDonald, PhD – Massachusetts General Hospital
Expanded HD (now HTT) CAG trinucleotide repeat alleles trigger the HD pathogenic process, in a dominant CAG length dependent fashion, and predict the eventual onset of HD symptoms. Furthermore, the CAG repeat is polymorphic in the population and there is now considerable molecular and biochemical evidence that the repeat acts as a quantitative functional polymorphism even through the normal allele range. A parsimonious explanation is an impact of the CAG-encoded polyglutamine tract on huntingtin that affects its structural properties, functional activity, subcellular location or other parameter. To uncover the pathogenic trigger mechanism and its immediate consequences, we employ a strategy that couples global phenotyping methods to the HD genetic criteria (true dominance, progressivity with CAG size, striatal specificity). An observation that fully conforms to these criteria was evidence, from Hdh CAG knock-in mice, for distinct conformational isoforms of full-length huntingtin, displaying distinct subsets of antibody epitopes, which appeared to be altered in location within striatal neurons early in the disease process. Now, application of the genetic approach to the discovery of the structural properties and posttranslational modifications of human huntingtin, using biophysical and mass spectrometry analysis of a panel of recombinant full-length proteins, has revealed that increasing polyglutamine tract length yields alternate patterns of phosphorylation encompassing more than a dozen sites across the entire protein, consistent with the progressive impact of the polyglutamine tract on huntingtin’s continuous α-helical structure. Indeed, these patterns were not found with huntingtin sub-domains. Moreover, global de-phosphorylation, like increased polyglutamine tract length, enhanced recombinant huntingtin activity in an in vitro assay system. Thus, structural conformers marked by alternate phosphorylation patterns may offer a posttranslational mechanism for polyglutamine tract modulation of huntingtin function, subcellular location or other property that may trigger pathogenesis. This view strongly urges PTM-based therapeutic approaches that target conformational isoforms of huntingtin, marked by distinct sets of coordinately altered phosphor-epitopes, in a manner designed to alleviate or circumvent the impact of the polyglutamine tract on huntingtin’s continuous α-helical structure.
Advances in RNAi Therapies for Huntington’s Disease
Beverly L. Davidson, PhD – University of Iowa
To date, a therapy for the neurodegenerative disorder, Huntington’s disease (HD), remains elusive. HD is characterized by cell loss in the basal ganglia, with particular damage to the putamen, an area of the brain responsible for initiating and refining motor movements. Accordingly, patients are plagued by a hyperkinetic disorder characterized by involuntary movements of their arms and legs. RNA interference (RNAi) has emerged as a candidate approach to treat HD. However, the safety of RNAi-based therapies remains a concern. High levels of exogenously supplied RNAi substrates can induce toxicity by saturating the endogenous RNAi machinery. Also, inhibitory RNAs have the potential to bind to and regulate unintended mRNA targets, an effect known as off-target gene silencing. Off-targeting primarily occurs when the seed region (nucleotides 2-8 of the small RNA) pairs with sequences within 3’ UTRs of unintended mRNAs and directs translational repression of those transcripts. To address these concerns, we undertook methods to improve the safety of RNAi expression vectors for in vivo applications. Using our recently developed siRNA design algorithm, we generated a panel of novel artificial miRNAs which upon processing would yield human huntingtin-specific siRNAs which preferentially load the antisense strand. Most importantly, these antisense strands contain seed sequences that are complementary to 6-mers that are present at low frequencies within all known human 3’UTRs. Microarray data show that these sequences have significantly attenuated (5 to 10-fold) off-target affects, a finding with wide-spread importance for therapeutic RNAi development. In studies in rodents and nonhuman primates, these sequences reduce overall HTT levels by ~50%. Gross and fine-motor testing in the NHPs, as well as histological and molecular readouts, demonstrate the safety of these sequences in vivo. These early data suggest that partial suppression of HTT in the primate putamen does not induce behavioral deficits or neuropathology, an important pre-requisite as we move this technology to clinical testing.
Antisense Based Therapeutics for the Treatment of Huntington’s Disease
C. Frank Bennett, PhD – Isis Pharmaceuticals
Antisense oligonucleotides, which are designed to bind to and modulate the function of protein coding and noncoding RNAs in a sequence specific manner, are being developed for the treatment of a broad range of diseases. An emerging area for antisense based drugs is for the treatment of neurodegenerative diseases. Two antisense drugs are currently in clinical trials for the treatment of amyotrophic lateral sclerosis and spinal muscular atrophy, and additional antisense drugs are in late stage research. We have demonstrated that antisense oligonucleotides distribute broadly in CNS tissues following intrathecal delivery and are well tolerated in rodents and non-human primates. Isis and our collaborators are pursuing three different antisense strategies for treating Huntington’s disease. The most advance strategy is targeting both the CAG expanded and wild-type huntingtin transcripts with RNase H oligonucleotides, which we have demonstrated huntingtin lowering in CNS tissues of non-human primates following intrathecal delivery. A second approach is to selective target single nucleotide polymorphisms (SNPs) that co-segregate with the CAG expansion using novel oligonucleotide designs that also recruit RNase H. A third approach is to selectively target the CAG repetitive sequence using oligonucleotides that work through a non-RNase H mechanism. The attributes and limitations of each approach will be discussed.
Zinc Finger Proteins: New Possibilities for HD Research and Therapy
Presentation not available
Steve Zhang, PhD – Sangamo BioSciences
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by CAGtrinucleotide repeat expansion in the first exon of the Huntingtin (Htt) gene. Repeat lengths of 35 or fewer CAGs are normal and have no associated pathophysiology, while those of 40 or more lead to HD with 100% penetrance, with longer repeat lengths correlating with earlier disease onset. The degeneration process primarily affects the basal ganglia and cerebral cortex, and the disease is characterized by a progressively worsening chorea, as well as cognitive and psychiatric dysfunctions. While neither the precise function of wild-type Htt protein nor the mechanism by which mutant Htt (which contains an expanded polyglutamine stretch) in HD pathogenesis is fully understood, results from rodent models of HD demonstrate that reducing mutant Htt levels, can prevent or delay disease onset. Thus, strategies that selectively reduce the expression of mutant and disease causing form of Htt represent the ideal therapeutic approach. Engineered zinc finger protein transcription factors (ZFP TF) can be designed to up- or down-regulate gene expression with exceptional specificity. Acting at the DNA level these factors turn virtually any gene into a potential drug target – a feature of particular significance for HD, where a genetic signature of disease has been identified that has thus far evaded classical small molecule drug intervention. This presentation will review recent data demonstrating that ZFP TFs can be designed to control Htt gene expression in both mouse and human cells. Moreover, data demonstrating the selective regulation of the mutant Htt allele will be presented. Together these results support the development of designer ZFPs for HD as (i) transcription factors (ZFP TFs) for controlling gene expression; and (ii) nucleases (ZFNs) for precision genome editing.
Use of Biomarkers to Bridge Preclinical and Clinical Studies of Huntingtin Suppression by RNAi Therapeutics
William F. Kaemmerer, PhD – Medtronic, Inc.
Reducing mutant Htt expression is a promising therapeutic strategy for the treatment of Huntington’s disease (HD), and RNA interference has emerged as a powerful approach for achieving this suppression. We have selected a potent small interfering RNA (siRNA), designed to suppress both mutant and wildtype Htt, as a clinical candidate. This siRNA will be administered to the CNS by direct infusion into the putamen with convection-enhanced delivery. Identification of biomarkers that provide read-outs of Htt lowering in the striatum and are feasible for clinical use, especially in early clinical trials, may greatly facilitate the clinical development of such a therapy. Candidate biomarkers include those that can be measured in the cerebrospinal fluid or peripheral blood with molecular methods, or in the brain employing a variety of imaging methods. Considerations around the relationship of these candidate biomarkers to disease progression in HD, and the status of the technical development of assays for detection of these biomarkers will be discussed. Finally, the use of transgenic rodent models of HD to assess the response of candidate biomarkers to Htt lowering will be critical, and will also be addressed. Continued substantial efforts will be essential in identifying biomarkers as early read-outs of Htt lowering, as we move this therapeutic approach towards the clinic.
Silencing Sheep – Exploring Safety and Efficacy of Viral Based Gene Therapy in a Large Animal Model
Presentation not yet available
Neil Aronin, MD – University of Massachusetts School of Medicine
Use of adeno-associated viral (AAV) delivery of small RNAs has had an auspicious start. In mice and non-human primates, AAV-shRNA or AAV-artificial miRNA has good spread and silences huntingtin mRNA. There are gaps in our knowledge of AAV administration to brain. Not established are the best AAV serotypes to gain widespread distribution in brain without neuronal damage or gliosis. Is single stranded AAV better suited and safer than double stranded AAV to deliver small RNAs in brain? Ensuring consistent, reproducible and robust expression of AAV-shRNAmir has high therapeutic value, but needs study. This discussion will consider AAV spread, safety, neuronal uptake, fidelity of small RNA expression in wild type sheep, and prospective studies in transgenic HD sheep. Supported by NIH, CHDI, Lundbeck Pharma.
Phosphodiesterases (PDEs) as CNS Targets
Christopher Schmidt, PhD – Pfizer, Inc.
The robust expression of PDEs in the brain makes these enzymes particularly attractive targets for CNS indications. We have developed highly selective, brain penetrant inhibitors for PDE10A, PDE9 and PDE2 with both PDE10A and PDE9 compounds advancing into phase 2 trials. PDE10A is a dual substrate enzyme highly expressed in the striatum where it regulates the sensitivity of medium spiny neurons to glutamatergic input. PDE10A inhibitors increase activity in both cyclic nucleotide signaling cascades as well as in the MAP kinase pathway and are extremely potent activators of striatal gene transcription. At the behavioral level, PDE10A inhibitors are effective in animal models predictive of antipsychotic activity. PDE9 is selective for cGMP and is expressed at low levels throughout the brain. Genetic or pharmacological inactivation of PDE9 results in elevations of cGMP in multiple brain regions and CSF. Consistent with it broad distribution, inhibition of PDE9 improves activity in a variety of models of monoaminergic and cholinergic dysfunction including amphetamine-disrupted auditory gating, 5HT2A agonist-mediated stereotypic behavior and scopolamine-disrupted spatial memory. PDE2A is a cGMP-activated, dual substrate PDE believed to function as an effector of nitric oxide signaling. Although highly expressed in projection neurons of the rat cortex and hippocampus, PDE2A protein is restricted to axons and terminals. Striatal PDE2A also exhibits a preference for efferent projections. Pharmacological inhibition of PDE2A increases regional concentrations of cGMP in rodent brain but may also regulate cAMP signaling by G(s) coupled receptors such as the D1 dopamine receptor. PDE2A inhibitors have been reported to improve performance in animal models of object and social recognition.
In collaboration with CHDI, we have recently demonstrated that inhibitors of PDE10A, PDE9 and PDE2A are effective in reversing or preventing elements of corticostriatal dysfunction in transgenic models of Huntington’s disease. We are now pursuing a strategy to evaluate these targets in additional preclinical models as well as in early patient studies using imaging and physiological outcome markers with the goal of progressing at least one of the inhibitors into a POC trial in the near future.
cGMP-hydrolyzing PDEs are Therapeutics Targets for Huntington’s Disease
Vahri Beaumont, PhD – CHDI
The contribution of impaired cyclic AMP (cAMP) signaling to altered synaptic plasticity and transcriptional dysregulation observed in Huntington’s disease (HD) is well described. Conversely, little attention has been paid to cyclic GMP (cGMP) signaling pathways in HD, despite a report of improvement in behavior and survival in the R6/2 HD mouse model following administration of TP- 10, a PDE10 inhibitor which elevates both cAMP and cGMP. In collaboration with Pfizer, CHDI has assessed the role of modulation of cGMP signaling via PDE inhibition as a therapeutic strategy, focusing especially on neuronal and synaptic assessment of HD models ex vivo and in vivo.
In corticostriatal slices taken from symptomatic R6/2 and Q175 knock-in mice, potent and selective inhibitors of dual cAMP/cGMP hydrolyzing PDEs PDE10A and PDE2, and an inhibitor of the cGMP-selective PDE9A, reversed multiple parameters of aberrant medium spiny neuron excitability, improved glutamatergic transmission in corticostriatal synapses and fully reversed deficits in hippocampal CA3-CA1 long term potentiation (LTP) in R6/2 mice. The hippocampal effects were not seen with cAMP-selective PDE4 inhibitors. In these acute experiments, all cGMP hydrolyzing PDE inhibitors were equally effective.
Additional evaluation of PDE9 and PDE10 inhibitors in vivo support their therapeutic utility in treating HD symptoms. Improvement of indirect pathway basal ganglia output in vivo was shown in a third model, the full length Htt transgenic BACHD rat, following acute systemic PDE10 inhibition. Further, following chronic dosing of Q175 mice with selective PDE10 and PDE9 inhibitors, we provide evidence of a sustained functional improvement in HD affected circuitry, with differential but complementary effects of the two inhibitors observed.
These data suggest that enhancing cGMP signaling can rectify neuronal and synaptic dysfunction in HD rodent models, in a way that would be expected to modulate striatally-driven thalamocortical circuit disturbances underlying hyperkinesia. CHDI’s focus is to aid the advancement of these PDE10 and PDE9 inhibitors to clinical trials for HD. An overview of our results to date and future plans in collaboration with Pfizer towards a clinical trajectory will be presented.
Neuropharmacology of Novel Kynurenine 3-monooxygenase Inhibitors in Animal Models of Huntington’s Disease
Ladislav Mrzljak, MD, PhD – CHDI
Metabolites of kynurenine pathway (KP) are implicated in the pathophysiology of neurodegenerative disorders including Huntington’s disease (HD). Kynurenine 3- monooxygenase (KMO) plays an important role in the KP since its inhibition leads to the increase of neuroactive metabolite kynurenic acid (KYNA) and decreases levels of the presumed neurotoxic metabolites 3-hydoxykynurenine (3- HK) and quinolinic acid (QA) in the brain. Inhibition of KMO with poor BBB penetrant compounds leads to the increase of kynurenine (KYN) in the blood and peripheral tissues. KYN crosses the BBB and is consequently converted to the KYNA by the enzyme kynurenine aminotransferase (KAT II) in brain astrocytes. Recently, it was demonstrated that peripheral inhibition of KMO with small molecule JM6 or KYNA up-regulation by means of KYNA analog have a neuroprotective effect in rodent models of HD. To test the hypothesis on whether the increase of KYNA in the brain through the peripheral inhibition of KMO leads to neuroprotective, anti-neuroinflammatory or beneficial synaptic effects, CHDI has developed highly potent KMO inhibitors with limited brain exposure including lead compound CHDI-00340246. CHDI-00340246 dose-dependently elevates the levels of kynurenine, AA and KYNA in the brain in rodent models of HD (BACHD rat and Q175 mice) and in WT animals. Neurophysiological slice experiments demonstrated that CHDI-00340246 restores normal excitability of striatal medium spiny neurons, and rescues hippocampal long term potentiation deficits when acutely administered to slices derived from HD mouse models. Despite the current belief in the field that neuroprotective effects of KMO inhibitors and KYNA are achieved in part through attenuation of glutamate (Glu) and dopamine (DA) release in the striatum, neurochemical analysis with multiple KMO inhibitors has failed to confirm their effects on Glu and DA release. Future and ongoing efficacy experiments to demonstrate potential beneficial effects of KMO inhibitors in rodent HD models using neurophysiological, molecular, and behavioral and neuroimaging endpoints will be discussed.
Selective mGluR5 Antagonists as Therapies for Movement and Neurodevelopmental Disorders
Presentation not yet available
Graeme Bilbe, PhD – Novartis
mGluR5 antagonists have been pursued as promising therapies for a variety of neurological disorders. I will describe the identification, pre-clinical characterization and early clinical development of AFQ056/Mavoglurant, a structurally novel, orally active, non-competitive mGlu5 receptor antagonist. Mavoglurant was discovered by chemical derivatization of a lead compound identified by HTS campaign. Its mode of action is by non-competitive inhibition of glutamate-induced activation of the human mGlu5 receptor. It is devoid of activity at human mGlu1, -2, -4, -7 receptors, the human GABAB receptor and a battery of more than 70 CNS-relevant receptors. In vivo, Mavoglurant has efficacy in a variety of animal assays for diseases such as Parkinson’s Disease, Fragile X Mental Retardation Syndrome, and gastro-esophageal reflux disorder. Further, Mavoglurant proved efficacious in clinical trials in patients with these diseases. Additional studies with Mavoglurant and other mGluR5 antagonists are underway in patients suffering from depression, chronic pain, dystonia and Huntington’s disease.
A Refreshing Look at Huntington’s Disease Clinical Trials
Cristina Sampaio, MD, PhD – CHDI
Clinical trials fail for a variety of reasons: 1) the intervention is not effective; 2) the effective dose is not properly defined or the likely efficacious dose cannot be administered due to safety concerns (or worse, untimely development planning) ; 3) the effect size is inadequate, and the therapeutic approach will require a synergistic paradigm; 4) outcomes are poorly chosen; 5) trial participant cohort is too small; 6) appropriate target population is not defined; 7) poor trial execution; 8) placebo effect; 9) random effect (chance). They are experiments after all!
However, under various circumstances many clinical trials in HD have been initiated when it was known a priori that they could not possibly succeed, unless an effect with a very low probability occurred. Even so, such trials may have provided valuable clinical knowledge, but at a motivational and emotional cost to the research participants involved and their families.
It is now time for the HD field to conduct well-defined, relatively small, tailored clinical trials of therapeutic candidates, taking in lessons from other relevant genetic and neurodegenerative disorders. Traditionally, the HD field has often followed advances and practices in the neurodegenerative field, and clearly there are currently interesting developments in trials for very early (preclinical) Alzheimer’s disease that can inform the HD field. However, advances in the treatment of genetic disorders, such as the stepwise successes with RNA interference treatment for the X-linked disease Duchenne muscular dystrophy, can pave the way for HD clinical trials too. Furthermore, the small definitive trials of substitutive therapies, such as those for Gaucher disease, should also be food for thought.
Clinical trials should be creative and adapted to the specific goals with demonstration of relevant benefits commensurable to the risks, rather than being a standardized deliverable for regulatory authorities with whom, according to some, no frank dialogue is deemed possible. We live in exciting times shared by all partners – clinicians, scientists, politicians, patients and families. The process of developing a successful clinical trial is a partnership among these shareholders.
TRACK-HD: Final Analysis and a New Beginning
Sarah Tabrizi, MD, PhD – University College London
Since 2008 TRACK-HD has chronicled the earliest stages of the neurodegenerative disease processes in premanifest and mild to moderately symptomatic individuals who carry the Huntington’s disease expansion mutation. TRACK-HD was designed to observe natural disease progression in premanifest and early stage HD with the aim to establish sensitive and specific clinical and biological markers of disease progression, which could serve as potential outcome measures in future disease modifying therapeutic trials.
We recently reported effect sizes for potential endpoints for clinical trials in early stage Huntington’s disease over 24 months. We also presented sample size calculations based on these effect sizes. These 24 month data will be presented in this talk in more detail, including insights into what we have learnt about Huntington’s disease progression in both the premanifest and early stages of the disease. The 36 month data from the TRACK-HD study is currently being analyzed, and some of that new data will also be presented.
Finally I will introduce our new study – Track-On HD – which aims to identify neural compensatory networks that may occur in the premanifest phase of neurodegeneration in HD. This study will test the hypothesis that functional reorganisation of networks may precede widespread structural loss in premanifest HD, and that symptom onset may reflect a decompensation of these neural compensatory networks. Track-On HD is due to start in March 2012 and an overview of the new study will be presented during this talk.
Huntington’s Disease Diagnosis: Moving from an Event to a Spectrum
Mark Guttman, MD, FRCPC – Center for Movement Disorders
Individuals at risk for HD are given a clinical diagnosis of the disease only after they have developed motor abnormalities. Clinicians, families and patients are aware that changes occur in many nonmotor domains, including cognition and behaviour, years before the motor-dependent clinical diagnosis is made in some individuals.
Longitudinal observational studies have identified that there are many changes that occur before the clinical diagnosis of HD. Researchers postulate that these changes should be considered part of the pathological process of HD. Since these changes have occurred before clinical diagnosis using motor criteria, to date it has been difficult to categorize them and to determine their relevance when planning clinical trials and dealing with regulatory agencies. Using the concept of a patient converting from a presymptomatic state to being diagnosed with HD as an endpoint of clinical trials creates considerable methodological problems. Furthermore, if this is to be the only focus of disease modifying research in HD, we will lose the opportunity to modify the underlying disease process in the earliest stages of the condition.
We propose a different conception of HD as the spectrum of clinical manifestations that includes the earliest symptoms as part of HD even before diagnosis by current diagnostic criteria. The paradigm shift is to change the focus of clinical research from the concept of preventing the transition of subjects from a “preclinical” to “diagnosed with HD” state to altering the progression of HD throughout the entire spectrum of the disease.
Changing Prevalence of HD: Altered Demographics and its Implications
Michael Hayden, MD, PhD – University of British Columbia
Increased longevity is significantly altering the demographics of many societies. The proportion of persons over 65 is around 7%. This proportion will double in the next 30 years to 14%, with close to 75% in developed countries such as the USA, Canada and Australia.
Currently HD is viewed as a disease of mid adult life affecting approximately 7.5 per 100,000. Disease onset is strongly influenced by age and CAG length. Current estimates of prevalence in British Columbia indicate that there has been a serious underestimate of disease prevalence. Furthermore an increasing number of persons greater than 60 are presenting for predictive testing based on development of signs of the disease in an elderly relative. A CAG length of 27-35 is seen in approximately 5% of the general population. These persons represent a source for alleles in the affected range. A CAG of 36-39 is present in approximately 0.25-0.5% (1 in 400/200) in the general population. The cumulative likelihood of developing HD by 75 for a person with a CAG of 36 is approximately 66% and over 90% for someone with a CAG of 39. With increasing longevity, there will be an increase in persons with late onset HD and the demographics of persons affected with HD will shift from a rare disease of midlife to a more common disease of the elderly.
A detailed assessment of CAG size in different populations will allow more focused estimates of predicted prevalence and needs for services and support in different parts of the world. In addition detailed haplotype analysis of chromosomes carrying the expanded CAG will allow for personalised intervention for allele specific therapeutic approaches.